Potato Proteinase Inhibitor Research Articles

GhWRKY75 positively regulates GhPR6-5b via binding to a W-box TTGAC (C/T) to orchestrate cotton resistance to Verticillium dahliae

Verticillium dahliae is an important fungal pathogen affecting cotton yield and quality. Therefore, the mining of V. dahlia-resistance genes is urgently needed. Proteases and protease inhibitors play crucial roles in plant defense responses. However, the functions and regulatory mechanisms of the protease inhibitor PR6 gene family remain largely unknown. This study provides a comprehensive analysis of the PR6 gene family in the cotton genome. We performed genome-wide identification and functional characterization of the cotton GhPR6 gene family, which belongs to the potato protease inhibitor I family of inhibitors. Thirty-nine PR6s were identified in Gossypium arboreum, G. raimondii, G. barbadense, and G. hirsutum, and they were clustered into four groups. Based on the analysis of pathogen-induced and Ghlmm transcriptome data, GhPR6-5b was identified as the key gene for V. dahliae resistance. Virus-induced gene silencing experiments revealed that cotton was more sensitive to V. dahliae V991 after PR6-5b silencing. The present study established that GhWRKY75 plays an important role in resistance to Verticillium wilt in cotton by positively regulating GhPR6-5b expression by directly binding to the W-box TTGAC(T/C). Our findings established that GhWRKY75 is a potential candidate for improving cotton resistance to V. dahliae, and provide primary information for further investigations and the development of specific strategies to bolster the defense mechanisms of cotton against V. dahliae.

Plant-Produced Therapeutic Crizanlizumab Monoclonal Antibody Binds P-Selectin to Alleviate Vaso-occlusive Pain Crises in Sickle Cell Disease.

Sickle Cell Disease (SCD) is a severe genetic disorder causing vascular occlusion and pain by upregulating the adhesion molecule P-selectin on endothelial cells and platelets. It primarily affects infants and children, causing chronic pain, circulatory problems, organ damage, and complications. Thus, effective treatment and management are crucial to reduce SCD-related risks. Anti-P-selectin antibody Crizanlizumab (Crimab) has been used to treat SCD. In this study, the heavy and light chain (HC and LC) genes of anti-P-Selectin antibody Crimab were cloned into a plant expression binary vector. The HC gene was under control of the duplicated 35S promoter and nopaline synthase (NOS) terminator, whereas the LC gene was under control of the potato proteinase inhibitor II (PIN2) promoter and PIN2 terminator. Agrobacterium tumefaciens LBA4404 was used to transfer the genes into the tobacco (Nicotiana tabacum cv. Xanthi) plant. In plants the genomic PCR and western blot confirmed gene presence and expression of HC and LC Crimab proteins in the plant, respectively. Crimab was successfully purified from transgenic plant leaf using protein A affinity chromatography. In ELISA, plant-derived Crimab (CrimabP) had similar binding activity to P-selectin compared to mammalian-derived Crimab (CrimabM). In surface plasmon resonance, the KD (dissociation binding constant) and response unit values were lower and higher than CrimabP, respectively. Taken together, these results demonstrate that the transgenic plant can be applied to produce biofunctional therapeutic monoclonal antibody.

Novel high-yield potato protease inhibitor panels block a wide array of proteases involved in viral infection and crucial tissue damage

Viruses critically rely on various proteases to ensure host cell entry and replication. In response to viral infection, the host will induce acute tissue inflammation pulled by granulocytes. Upon hyperactivation, neutrophil granulocytes may cause undue tissue damage through proteolytic degradation of the extracellular matrix. Here, we assess the potential of protease inhibitors (PI) derived from potatoes in inhibiting viral infection and reducing tissue damage. The original full spectrum of potato PI was developed into five fractions by means of chromatography and hydrolysis. Individual fractions showed varying inhibitory efficacy towards a panel of proteases including trypsin, chymotrypsin, ACE2, elastase, and cathepsins B and L. The fractions did not interfere with SARS-CoV-2 infection of Vero E6 cells in vitro. Importantly, two of the fractions fully inhibited elastin-degrading activity of complete primary human neutrophil degranulate. These data warrant further development of potato PI fractions for biomedical purposes, including tissue damage crucial to SARS-CoV-2 pathogenesis.Key messagesProtease inhibitor fractions from potato differentially inhibit a series of human proteases involved in viral replication and in tissue damage by overshoot inflammation.Protease inhibition of cell surface receptors such as ACE2 does not prevent virus infection of Vero cells in vitro.Protease inhibitors derived from potato can fully inhibit elastin-degrading primary human neutrophil proteases.Protease inhibitor fractions can be produced at high scale (hundreds of thousands of kilograms, i.e., tons) allowing economically feasible application in lower and higher income countries.

Functional Food Based on Potato.

Potato (Solanum tuberosum L.) has gradually become a stable food worldwide since it can be a practical nutritional supplement and antioxidant as well as an energy provider for human beings. Financially and nutritionally, the cultivation and utility of potatoes is worthy of attention from the world. Exploring the functionality and maximizing the utilization of its component parts as well as developing new products based on the potato is still an ongoing issue. To maximize the benefits of potato and induce new high-value products while avoiding unfavorable properties of the crop has been a growing trend in food and medical areas. This review intends to summarize the factors that influence changes in the key functional components of potatoes and to discuss the focus of referenced literature which may require further research efforts. Next, it summarizes the application of the latest commercial products and potential value of components existing in potato. In particular, there are several main tasks for future potato research: preparing starchy foods for special groups of people and developing fiber-rich products to supply dietary fiber intake, manufacturing bio-friendly and specific design films/coatings in the packaging industry, extracting bioactive proteins and potato protease inhibitors with high biological activity, and continuing to build and examine the health benefits of new commercial products based on potato protein. Notably, preservation methods play a key role in the phytochemical content left in foods, and potato performs superiorly to many common vegetables when meeting the demands of daily mineral intake and alleviating mineral deficiencies.

Potato protease inhibitors, a functional food material with antioxidant and anticancer potential

Potato protease inhibitors (PPIs), as the main component of potato protein isolate, have good safety, nutrition and great market potential. The antioxidant and anticancer properties of PPIs were evaluated with cell-based biological assays. The results showed that when the concentration of PPIs was 5 mg/mL, the peroxyl radical scavenging value was (2119 ± 204) mg VCE/100 g, and the cellular antioxidant activity values were (45.83 ± 3.5) (no PBS wash) and (33.25 ± 4.4) µmol QE/100 g (PBS wash). Cells pretreated with PPIs could significantly prevent the oxidative damage induced by H2O2, inhibit the morphological changes of cells and maintain the integrity. Furthermore, PPIs had selective anti-proliferative effects on GIST882 cells (IC50 = (10.53 ± 3.87) mg/mL) and demonstrated potent inhibition of the growth, migration and invasion of cancer cells. These findings provide a scientific basis for PPIs as promising candidates for functional foods to aid in the prevention of oxidative damage and cancer.

Potato protease inhibitor II prevents obesity by inducing browning of white adipose tissue in mice via β3 adrenergic receptor signaling pathway.

There are currently few effective and safe pharmacologic means for inducing beige adipogenesis in humans. This study highlights the role of potato protease inhibitor II (PPI II) in regulating the browning of adipose tissue. The in vitro results showed that PPI II increased the expression of the uncoupling protein 1 (UCP1) protein and gene and beige-specific genes, including Cd137, Cited1, Tbx1, and Tmem26 in vitro. PPI II treatment for three months in diet-induced obesity mice increased the levels of the UCP1 protein in white adipose tissue, causing elevated energy expenditure, thus preventing obesity and improving glucose tolerance. Mechanistic studies further revealed that PPI II regulated the abundance and activity of β3 adrenergic receptor (β3 -AR) in white adipocytes. Chemical-inhibition experiments revealed the crucial role of β3 -AR-dependent protein kinase A (PKA)-p38 kinase (p38)/extracellular signal-related kinase1/2 (ERK1/2) signaling in PPI II-mediated browning program of white adipose tissues. In summary, our findings highlight the role of PPI II in beige adipocyte differentiation and thermogenesis and provide new insights into its use in preventing obesity.

Oxidative cross-linking of potato proteins by fungal laccases: Reaction kinetics and effects on the structural and functional properties

Aiming at developing biocatalytic approach to modulate potato proteins functionalities, laccase-catalyzed oxidative cross-linking of potato proteins was investigated in terms of enzyme kinetics, reaction time course and product structural-functional properties. The catalytic efficiency (kcat/Km, mM−1S−1) of fungal laccases in oxidizing patatin-enriched potato protein (PAT, 0.010–0.748) was higher than that of potato protease inhibitors (PIs, 0.008–0.184). While PIs formed more efficiently oxidative cross-linked products. Cross-linking of potato proteins were achieved by laccase alone or by laccase-ferulic acid system; ferulic acid contributed to higher cross-linking extent and to the antioxidant activity of modified proteins. Potato proteins exhibited similar or slightly enhanced solubility upon cross-linking, except that highly cross-linked PIs (cross-linking extent >30%) obtained from Coriolus hirsutus laccase (LacCh)-treatments showed reduced solubility. General improvement in the emulsification property of potato proteins was observed upon cross-linking. Cross-linked potato proteins from LacCh-6 h treatment showed good foaming property; these proteins exhibited enhanced molecular flexibility and the characteristics of mild cross-linking extent (9.6–17.1%) with the accumulation of moderate molecular weight fraction (PAT:60–80 kDa, PIs:30–40 kDa). This study will lay foundations for the exploitation of potato proteins as functional ingredients. Industrial relevanceThe exploitation of novel proteins as functional ingredients is needed in order to meet the high demands for natural, healthy-oriented and sustainable food products. Laccase-catalyzed protein cross-linking is a promising green technology for tailoring the functionality of protein ingredients and achieving the desired textural/rheological quality of final products. The current study demonstrated the effectiveness of this enzymatic approach to enhance foaming and emulsifying properties of potato proteins. This approach can be adopted to modulate the functionality of other plant proteins and to develop plant proteins-based functional ingredients for various applications including stable delivery system, fat-replacing biopolymer matrix, meat analog/vegan products and gluten-replacement formulations.

Preparation and antioxidative stability of the potato protease inhibitors (PPIs) from potato starch waste-water

Potato protease inhibitors (PPIs) isolate, a major recovery protein from starch waste-water, possesses promising bio-activity and attracts growing attention. In this study, PPIs was extracted with a simple and cost-saving procedure, coupling salting out precipitation and isoelectric point precipitation. Antioxidant activity of PPIs isolate was evaluated by different methods. It displayed the good capacity for scavenging toward ABTS radicals (IC50 = 0.02 mg/mL) and inhibition of lipid peroxidation (IC50 = 0.30 mg/mL), especially, the IC50 was lower than that of ascorbic acid (VC, IC50 = 0.44 mg/mL) and tocopherol (VE, IC50 = 0.34 mg/mL). Moreover, the ABTS radicals scavenging ability remained high level at 50 °C and a pH range of 6–11; the inhibition of lipid peroxidation was stable at 25 °C and a pH range of 2–8. And all the antioxidant activities possessed satisfactory stability under ultrahigh-pressure condition. The results present great potential of PPIs as a high-quality protein with antioxidant function and to be used in functional foods.

Protein-enriched fiber vegan meal promote satiety and suppress food intake in rats.

Dietary preferences were closely associated with the pathogenesis of numbers of metabolic disorders, in particularly, obesity. Dietary fiber was shown to be capable of preventing weight gain and excessive food intake mainly through stimulating chewing and saliva secretion, and promote satiety signals. In this study, we characterized the "Vitamin World® Vegan Meal" Formula of Feihe, a novel protein‐enriched fiber dietary supplement contained potato protease inhibitor II (PI2) that developed. And we demonstrated that this particular fiber formula was effective in preventing weight gain, increasing satiety signals, and reducing food intake in rats in a dosage‐dependent manner. Our study provides lines of evidence and would further bolster the use of this nutritious vegan meal in regulating satiety and food intake in clinics.

Cysteine Protease Inhibitors Reduce Enzymatic Browning of Potato by Lowering the Accumulation of Free Amino Acids.

Enzymatic browning is a major issue affecting the quality of processed potato (Solanum tuberosum L.). To understand the molecular mechanism of browning, transcriptional analyses were performed by employing potatoes that differed in browning. Coexpression analysis indicated that 9 out of 15 upregulated genes in browning-less groups encoded for potato protease inhibitors (StPIs). In addition, gene otology analysis showed that the enriched terms were mainly involved in protease inhibitors. Overexpression of cysteine StPI 143 and StPI 146 individually reduced browning and lowered protease activities and tyrosine and total free amino acid (FAA) contents, but they could not decrease polyphenol oxidase activity. Moreover, supplementing exogenous tyrosine or total FAAs into transgenic potato mash to wild-type amounts promoted mash browning, browning with total FAAs, more than with tyrosine, resembling wild-type levels. These results implied that cysteine StPIs reduced browning via lowering the accumulation of FAAs in addition to tyrosine. Our findings have enriched the knowledge about the roles and mechanisms of protease inhibitors in regulating enzymatic browning of potato, which provide new ways for controlling potato browning.

The Effect of Potato Protease Inhibitor II on Gastrointestinal Hormones and Satiety in Humans During Weight Reduction.

ContextIt is questioned whether the potato protein protease inhibitor II (PI2) reduces appetite and exerts effects on the satiety hormone cholecystokinin (CCK).ObjectiveTo investigate PI2 impact on gastrointestinal hormones and appetite measures during weight reduction.DesignIn a randomized, placebo-controlled trial over 20 weeks, fifty-two overweight/obese participants (BMI 25.2–38.0 kg/m2) received a protein-rich diet (30%) adjusted to 500 kcal below their individual daily needs. Subjects ingested a capsule containing either PI2 (150 mg) or placebo twice daily 1 hr before lunch and dinner. At week 0 and week 10 participants joined breakfast test meals to determine CCK, GLP-1, ghrelin, leptin, glucose and insulin concentrations in a time course experimental manner. Appetite sensations were measured on test meal days and in week 4, 9, 14 and 19 using visual analogue scales.ResultsWeight loss at week 10 and 20 in the PI2 group was 4.3±3.1 kg and 5.6±4.1 kg, in the control group: 4.7±4.0 kg and 6.8±3.7 kg. A significant effect of PI2 on circulating CCK levels was observed at week 10. The other hormones were unaffected by PI2. At week 10, PI2 group subjects showed higher satiety and decreased desire to eat compared to placebo. During study duration, PI2 showed a significant impact on appetite ratings prior to lunch, one hour before dinner and just before dinner.ConclusionPI2 increased circulating CCK plasma levels during the diet intervention. Likewise, PI2 modulated appetite sensation from week 4 to 20. The study demonstrated that the PI2 can modulate a key satiety signal.

Coix lacryma-jobi chymotrypsin inhibitor displays antifungal activity

A novel chymotrypsin inhibitor, named ClCI, was purified from coix seed (Coix lacryma-jobi L.) by aqueous two-phase extraction, chymotrypsin–Sepharose 4B affinity chromatography and centrifugal ultrafiltration. ClCI was a 7.9 kDa competitive inhibitor with pI 6.54. The inhibition constants (Ki) for bovine pancreatic chymotrypsin and bacterial subtilisin were 1.27 × 10−10 M and 1.57 × 10−9 M respectively. ClCI had no inhibitory activity against bovine trypsin and porcine elastase. ClCI had wide pH stability and good heat resistance. It can maintain >90% inhibition activity against chymotrypsin at 20–80 °C for 1 h. The primary structure of ClCI was highly similar (57%–92%) to those of several inhibitors belonging to the Gramineae crop potato protease inhibitor- I superfamily and showed the typical sequence motif of the protease inhibitor of the seed storage protein group. ClCI (12.5 mg) inhibited mycelial growth of the phytopathogenic fungi Mycosphaerella melonis, Helminthosporium turcicum, Alternaria solani, Phytophthora capsici, Isariopsis griseola, and Colletotrichum gloeosporioides, and caused 89% inhibition of the proteases from spore germination of plant-pathogenic fungi. The results of the present study indicate that ClCI had biotechnological potential as an alternative agent to combat the important phytopathogenic fungi.

Antifungal and antimicrobial proteins and peptides of potato (Solanum tuberosum L.) tubers and their applications.

Potato proteins are well known for their nutritional, emulsifying, foaming, gel forming or antioxidant properties that all make from them valuable protein source for food industry. Antifungal, antimicrobial and also antiviral properties, described for potato proteins in the review, enrich the possibilities of potato protein usage. Potato proteins were divided into patatin, protease inhibitors and fraction of other proteins that also included, besides others, proteins involved in potato defence physiology. All these proteins groups provide proteins and peptides with antifungal and/or antimicrobial actions. Patatins, obtained from cultivars with resistance to Phytophthora infestans, were able to inhibit spore germination of this pathogen. Protease inhibitors represent the structurally heterogeneous group with broad range of antifungal and antimicrobial activities. Potato protease inhibitors I and II reduced the growth of Phytophthora infestans, Rhizoctonia solani and Botrytis cinerea or of the fungi of Fusarium genus. Members of Kunitz family (proteins Potide-G, AFP-J, Potamin-1 or PG-2) were able to inhibit serious pathogens such as Staphylococcus aureus, Listeria monocytogenes, Escherichia coli or Candida albicans. Potato snakins, defensins and pseudothionins are discussed for their ability to inhibit serious potato fungi as well as bacterial pathogens. Potato proteins with the ability to inhibit growth of pathogens were used for developing of pathogen-resistant transgenic plants for crop improvement. Incorporation of potato antifungal and antimicrobial proteins in feed and food products or food packages for elimination of hygienically risk pathogens brings new possibility of potato protein usage.

Proteomic characterization and antifungal activity of potato tuber proteins isolated from starch production waste under different temperature regimes.

Proteins were obtained from effluent of a starch manufacture by using different isolation temperatures (40, 60, 80, and 100°C). The proteins, remaining in effluent after treatment of potato juice at 80 and 100°C differed significantly in composition and in structural stability as well as in trypsin inhibitory and antifungal activities in comparison with the variants of 40 and 60°C. The protein samples of 80°C exhibited the highest antifungal activity and its average value of IC50 against five strains of two Fusarium species was determined in average at 0.18mgml-1. The 80°C protein samples consisted predominantly of low-molecular proteins (7-17kDa) identified as potato tuber protease inhibitors I and II. Predominantly, protease inhibitors II were identified for the protein samples obtained by 100°C and here we identified 7 spots in comparison with 12 identified for the 80°C samples. Samples of 40 and 60°C with low antifungal activities represent high variability of detected and identified proteins. We identified various representatives of aspartic, cysteine, and serine protease inhibitors in both types of samples. These samples also contained Kunitz-type protease inhibitors that were not found in the 80 and 100°C samples which documented thermal unstableness of Kunitz-type protease inhibitors. Functional stability at high temperatures and antifungal activity of isolated potato protease inhibitors I and II support the potential of this fraction usage in food, feed, pharmaceutical, or agricultural industry and offer new products for starch manufactures. At the same time, utilization of the stable protein fraction of waste deproteinized potato water promotes exploitation of potato starch production resources.

Molecular Cloning and Structure–Function Analysis of a Trypsin Inhibitor from Tartary Buckwheat and Its Application in Combating Phytopathogenic Fungi

Host plant protease inhibitors offer resistance to proteases from invading pathogens. Trypsin inhibitors (TIs), in particular, serve as protective agents against insect and pathogen attacks. In this study, we designed a pair of degenerate primers based on highly conserved motifs at the N- and C-termini of the TI from tartary buckwheat (Fagopyrum tataricum; Ft) to clone the central portion. Genomic walking was performed to isolate the 5′ and 3′ flanking regions of FtTI. We demonstrated the successful PCR amplification of a 644 bp portion of FtTI. The full-length DNA of FtTI contains a complete open reading frame of 264 bp, encoding 87 amino acids with a mass of approximately 9.5 kDa. The FtTI protein sequence was 49% identical and 67% similar to potato protease inhibitors. Site-directed mutagenesis identified the residues, Asp67 and Arg68, as crucial for the inhibitory activity of the FtTI. Recombinant and mutant FtTI inhibited both the hyphal growth and spore germination of Alternaria solani. The calculated 50% inhibitory concentrations of FtTI ranged from 5–100 μg mL−1 for spore germination and 1–50 μg mL−1 for fungal growth. Thus, recombinant FtTI may function in host resistance against a variety of fungal plant pathogens.

Recovery of Protease Inhibitors from Potato Fruit Water by Expanded Bed Adsorption Chromatography in Pilot Scale

The current study was conducted to investigate the recovery of native potato protein from potato fruit water (PFW) by expanded bed adsorption (EBA) chromatography. The eluted proteins were concentrated by ultrafiltration and spray-dried into powder. The SDS-PAGE showed that the recovered proteins were potato protease inhibitors (PPIs). The trypsin and chymotrypsin inhibitor activities of the recovered PPIs were 377.93 ± 8.22 and 12.90 ± 0.03 mg g−1 protein, respectively. The recovery yield of protease inhibitors was 74.88%. The glycoalkaloid assay showed that the recovered PPIs contained 30.31 ± 0.15 μg g−1 of α-chaconine and 92.77 ± 0.52 μg g−1 of α-solanine, and these values were much lower than those in potato protein concentrate (PPC) obtained by traditional thermal coagulation. The most abundant amino acid in the PPIs was serine. The results indicated that the EBA can be used to effectively recover native potato protein from PFW.

Potato protease inhibitor II suppresses postprandial appetite in healthy women: a randomized double-blind placebo-controlled trial

The effect of potato protease inhibitor II (PI2) on postprandial appetite was examined in a randomized double-blind placebo-controlled cross-over trial involving 44 healthy women. In separate test sessions, participants consumed a capsule containing placebo or potato extract standardized to 15 or 30 mg PI2 after overnight fasting. One hour later, a standard 390 kcal breakfast was served. At regular time points during the three-hour period after breakfast, appetite was measured by visual analog scales, and blood samples were collected for assay of cholecystokinin, insulin, and glucose. Compared with the placebo, consumption of 15 mg or 30 mg PI2 one hour prior to a standard breakfast meal resulted in significantly lower postprandial hunger, desire to eat, and prospective consumption, as well as significantly higher postprandial fullness. Consumption of 15 mg PI2 also resulted in significantly higher postprandial plasma levels of cholecystokinin compared with the placebo. No significant main effect of treatment was found on insulin and glucose. No adverse events were reported. Results from the study revealed that consumption of potato PI2 at the examined doses was well tolerated, suppressed subjective appetite in a dose-dependent manner, and increased plasma concentrations of cholecystokinin. Future studies are needed to evaluate the long-term effect of PI2 on body weight.

28 Days oral toxicity test of potato proteinase inhibitor II in swine

Proteinase inhibitor II (PI2), a compound derived from potato, is believed to help manage weight in humans when consumed as a dietary supplement. In this study, we evaluated the potential toxicity of PI2 in forty healthy swine weighing between 33 and 44 kg. The animals were treated daily for 28 days with oral doses of water, 300 mg, or 30 mg PI2. Additional control animals did not receive oral dosing, but were restricted in feed to the approximate amount consumed by animals treated with 300 mg PI2. Treatments did not significantly affect growth over the treatment period. However, significant effects were observed in some serum and hematologic indices, even though all values remained within normal physiological levels. At necropsy at the end of treatment, gross organ pathology and histopathology did not reveal microscopic lesions or structural changes due to treatments. Taken together, results indicate that oral doses of 30 mg or 300 mg of PI2 for 28 days are not associated with toxicity or adverse effects in swine.

<i>In vitro</i> antioxidant and anti-adipogenic effects of slendesta, standard potato extracts containing 5% protease inhibitor II

Background: The objective of the present study is to observe the anti-adipogenic effects of Slendesta (SLD), a standard potato protein extracts containing 5% potato protease inhibitor II (PI2) on the 3T3-L1 preadipocytes which are able to differentiate into mature adipocytes and accumulate lipids, as an obesity model with cytotoxicity and antioxidant effects.Materials and Methods: The cytotoxicity of SLD was observed against 3T3-L1 preadipocyte cell line by MTT assay, and also antiadipogenic effects were observed through lipid accumulation assay during 3T3-L1 differentiation as comparing with N-Acetyl-Lcysteine (NAC). In addition, antioxidant effects of SLD were detected by free radical scavenging capacity and superoxide dismutase (SOD)-like activity as comparing with ascorbic acid.Results: The SLD showed obvious cytotoxicity against 3T3-L1 pre-adipocyte cell line at higher concentrations, from 1.5 mg/ml for 72 h treatment, and the cytotoxic IC50 of SLD after 24, 48 and 72 h treatment times were detected as 10.11 ± 0.67, 5.71 ± 0.37 and 5.34 ± 0.21 mg/ml, respectively. The SLD also concentration-dependently inhibited the lipid accumulations formatted during 3T3-L1 cell differentiations. The adipogenic specific genes including PPARγ, C/EBPα, C/EBPβ and leptin were found to be reduced in SLD and NAC-treated cells compared to control cells. Furthermore, the SLD effectively showed DPPH radical scavenging activity (IC50 = 161.98 ± 64.65 μg/ml) and SOD-like effects (IC50 = 284.54 ± 54.47 μg/ml), and the cellular ROS was significantly inhibited in the SLD-treated cells compared to control cells.Conclusion: The results suggest that SLD effectively inhibit the differentiations of 3T3-L1 preadipose cell probably through antioxidant activities and direct cytotoxicity in case of higher concentration, along with satiety effects mediated by increases of circulating cholecystokinin. These findings are considered as direct evidences that SLD may serve as a predictable functional ingredient for obesity as an alternative therapy.Key words: Slendesta, potato protease inhibitor II, 3T3-L1 cell, cytotoxicity, anti-adipogenic effects, antioxidant effects.

IN VITRO ANTIOXIDANT AND ANTI-ADIPOGENIC EFFECTS OF SLENDESTATM, STANDARD POTATO EXTRACTS CONTAINS 5% PROTEASE INHIBITOR II

Background: The objective of present study is to observe the anti-adipogenic effects of SlenestaTM (SLD), a standard potato protein extracts containing 5% potato protease inhibitor II (PI2) on the 3T3-L1 preadipocytes which are able to differentiate into mature adipocytes and accumulate lipids, as an obesity model with cytotoxicity and antioxidant effects. 
 Materials and Methods: The cytotoxicity of SLD was observed against 3T3-L1 pre-adipocyte cell line by MTT assay, and also anti-adipogenic effects were observed through lipid accumulation assay during 3T3-L1 differentiation as comparing with N-Acetyl-L-cysteine (NAC). In addition, antioxidant effects of SLD were detected by free radical scavenging capacity and superoxide dismutase (SOD)-like activity as comparing with ascorbic acid. 
 Results: SLD showed obvious cytotoxicity against 3T3-L1 pre-adipocyte cell line at higher concentrations, from 1.5 mg/ml for 72 hrs treatment, and cytotoxic IC50 of SLD after 24, 48 and 72 hrs treatment times were detected as 10.11 ± 0.67, 5.71 ± 0.37 and 5.34 ± 0.21 mg/ml, respectively. SLD concentration-dependently inhibited the lipid accumulations formatted during 3T3-L1 cell differentiations, and also effectively showed DPPH radical scavenging activity (IC50 = 161.98 ± 64.65 μg/ml) and SOD-like effects (IC50 = 284.54 ± 54.47 μg/ml). 
 Conclusion: The results suggest that SLD effectively inhibited 3T3-L1 adipose cell differentiations may be through antioxidant activities and direct cytotoxicity in case of higher concentration, along with already known satiety effects mediated by increases of circulating cholecystokinin. These findings are considered as direct evidences that SLD may serve as a predictable functional ingredient for obesity as an alternative therapy.